The present invention pertains to biologically active, polypeptide fragments of mammalian bactericidal/permeability-increasing proteins and to methods for making and using said fragments.
Bactericidal/permeability-increasing protein (BPI) is a 50 to 60 Kd protein, isolated from the granules of mammalian polymorphonuclear leukocytes (PMN) which are blood cells that are essential in the defense against invading microorganisms in mammals. BPI occurs only in cells of the myeloid series of blood cells, is produced at the promyelocytic/myelocytic stage of differentiation and is located in the primary granules in these cells.
BPI is a potent cytotoxin (and therefore bactericidal agent) active against a broad range of gram negative bacterial species. BPI also inhibits endotoxin activity by neutralizing gram negative bacterial lipopolysaccharides (LPS). It exhibits a high degree of specificity in its cytotoxic effect: for example, 10-40 nM (0.5-2.0 micrograms) kill more than 90% of 10.sup.7 sensitive bacteria whereas 100-fold higher concentrations of BPI are non-toxic for other microorganisms and eukaryotic cells. All available evidence suggests that in the intact PMN and in chide leukocyte fractions, BPI is the principal oxygen-independent agent that is active against BPI-sensitive bacteria. Furthermore, its anti-LPS activity makes BPI a very good candidate for down-regulating responses triggered by LPS not only by halting bacterial proliferation but also by directly inhibiting the effects of released LPS. Endotoxins, i.e., complex lipopolysaccharides (LPS) are the major components of the outer envelope of gram negative bacteria, and account primarily for the most serious clinical consequences of gram negative bacterial infections. Young, L. S. in: Principles of Infectious Diseases (1990), Mandell, G. L., Douglas, R. D., and Bennett, J. E., eds., Churchill-Livingstone, New York, pp. 611-635. As little as subnanogram-to-ng/ml concentrations of these agents elicit in the host many cellular and extracellular responses, including the production and release of a diverse network of mediators such as activated Complement components, cytokines (e.g., tumor necrosis factor, interleukins), arachidonate metabolites, etc. Morrison, D. C. and Ryan, J. L. (1987) Ann. Rev. Med., 38:417-432. While these reactions must have evolved as part of essential host defenses, they may also become excessive and self-destructive. Young, (1990), supra; Morrison, (1987), supra; Beutler, B. and Cerami, A. (1986) Nature, 320:584-588, prompting many investigators to attempt to attenuate host responses to LPS. Recent reports indicate life-saving effects of monoclonal antibodies, directed against the biologically active lipid A portion of the LPS molecule, in Patients with gram negative bacteremia and sepsis. Ziegler, E., Fisher, C., Sprung, C., Straube, R., and Sadoff, J. (1990) Clin. Res., 38:304A, in accord with a primary role of endotoxin in the pathogenesis of gram negative sepsis and supporting the potential clinical usefulness of anti-LPS directed therapies.
Progress has also been made in the identification and characterization of endogenous LPS-binding proteins, produced by various cells and tissues. Ulevitch, R. J., Johnston, A. R., and Weinstein, D. B. (1981) J. Clin. Invest., 67:827-837; Munford, R. S., Andersen, J. M., and Dietschy, J. M. (1981) J. Clin. Invest., 68:1503-1513; Munford, R. S., and Hall, C. L. (1986) Science, 234:203-205; Tobias, P. S., Soldau, K., and Ulevitch, R. J. (1986) J. Exp. Med., 164:777-793; Roeder, D. J., Lei, M., and Morrison, D. C. (1989) Infect. and Immun., 57: 1054-1058, that may mediate and regulate the host's response to LPS. Among these proteins is a LPS-binding protein (LBP) that is produced and secreted by the liver and accumulates in plasma at least one hundred-fold higher than normal concentrations during the acute phase response that is triggered by LPS and many other irritants. Tobias, (1986), supra. In vitro LBP enhances the delivery of LPS and LPS-containing cells to macrophages and magnifies responses of macrophages and polymorphonuclear leukocytes (PMN) to LPS (Wright, S. D., Tobias, P. S., Ulevitch, R. J., and Ramos, R. A. (1989) J. Exp. Med., 170:1231-1241; Vosbeck, K., Tobias, P. S., Mueller, H., Allen, R. A., Arfors, K., Ulevitch, R. J., and Sklar L. A. (1990) J. Leuk. Biol., 47:97-104), suggesting that the function of LBP is to heighten the host's response to LPS. In contrast, a related LPS-binding protein present in PMN, the bactericidal/permeability-increasing protein (BPI; Tobias, P. S., Mathison, L. C., and Ulevitch, R. J. (1988) J. Biol. Chem., 263: 13479-13481 ), suppresses both endotoxin-mediated activation of procoagulant protease(s) in Limulus amebocyte lysates and upregulation of CR1 and CR3 receptors on the surface of PMN (Marra, M. N., Wilde, C. G., Griffith, J. E., Snable, J. L., and Scott, R. W. (1990) J. Immunol., 144:662-666).
BPI isolated from both human and rabbit PMN has been purified to homogeneity. The molecular, weight of human BPI is approximately 58,000 Daltons (58 kDa) and that of rabbit BPI is approximately 50 kDa. The amino acid composition of these two proteins is closely similar as is the amino acid sequence of their NH.sub.2 -terminal amino acid residues. Both proteins are highly basic, having an isoelectric point greater than 9.6.
The anti-microbial effects of BPI require attachment of the protein to the surface of the susceptible gram negative bacteria. Initial binding of BPI to target cells involves electrostatic interactions between the basic protein and the negatively charged sites on the lipopolysaccharides (LPS) on the properties of the bacterial outer membrane and leads to discrete alteration in the permeability properties of the bacterial membrane and activation of enzymes that degrade phospholipids and peptidoglycans. The final stage of action is the actual killing of the bacteria by an as yet unknown mechanism that involves damage to structural and functional elements associated with the cytoplasmic membrane. The closely similar amino acid composition and nearly identical bactericidal and membrane-perturbing properties of BPI purified from human and rabbit PMN suggest that this protein has been highly conserved during evolution and is an important member of the anti-bacterial arsenal of the mammalian PMN.
Due to its potent bactericidal action against gram negative bacteria and lack of cytotoxicity towards other microorganisms and eukaryotic cells, it is envisioned that BPI may be employed as a chemotherapeutic agent and/or as a model for the design of new antibiotic agents. The possibility has been raised that, as in the case with other cytotoxic proteins, the different functions of BPI, namely binding, envelope-altering and killing reside in different domains within the BPI molecule. Although BPI fragments, obtained by digestion of the holoproteins with the proteolytic enzyme elastase, have been reported (Weiss, J. et al., Clin. Res., 34:537A, 1986), the fragments tested remained associated under the non-denaturing conditions employed. No biological activity was ascribed to any isolated fragments. Moreover, antibodies directed against the holoprotein did not recognize these fragments under denaturing condition when analyzed using the well-known Western blotting procedure.
Therefore, in light of the above, there is a need in the art for biologically active peptide fragments of BPI for use as bactericidal/permeability increasing agents as well as therapeutic (e.g., antibacterial and antiendotoxin) agents. Such BPI fragments are also needed to provide structural information to direct the design of future generations of novel antimicrobial agents specific against gram negative bacteria and to be used as probes into the molecular organization of the multifunctional holo-BPI protein.